Electrogenic binding of ions at the cytoplasmic side of the Na+,K+-ATPase

Abstract

Abstract-Electrogenic binding of ions from the cytoplasmic side of the Na + ,K + ATPase has been studied by measurements of changes of the membrane capacitance and conductance triggered by a jump of pH or of the sodium ion concentration in the absence of ATP. The pH jumps were performed in experiments with membrane fragments containing purified Na + ,K + ATPase adsorbed to a bilayer lipid membrane (BLM). Protons were released in a sub millisecond time range from a photosensitive compound (caged H + ) triggered by a UV light flash. The sodium concentration jumps were carried out by a fast solution exchange in experi ments with membrane fragments attached to a solid supported membrane deposited on a gold electrode. The change of the membrane capacitance triggered by the pH jump depended on the sodium ion concentration. Potassium ions had a similar effect on the capacitance change triggered by a pH jump. The effects of these ions are explained by the their competition with protons in the binding sites on cytoplasmic side of the Na + ,K + ATPase. The approximation of the experimental data by a theoretical model yields the dissociation constants, K, and the cooperativity coefficients, n, of the binding sites for sodium ions (K = 2.7 mM, n = 2) and potassium ions (K = 1.7 mM, n = 2). In the presence of magnesium ions the apparent dissociation constants of sodium increased. A possible reason of the inhibition of sodium ion binding by magnesium ions can be an elec trostatic or conformational effect of magnesium ions bound to a separate site of the Na + ,K + ATPase close to the entrance to the sodium ion binding sites.

Binding of Potassium Ions Inside the Access Channel at the Cytoplasmic Side of Na+,K+-ATPase

“…The addition of KCl up to 10 mM did not affect this increase. After membrane fragments with Na + ,K + -ATPase were adsorbed to the BLM, the change of capacitance was larger, and the addition of KCl to the solution reduced this capacitance change, again similarly to that observed earlier [27].…”

“…Figure 1 shows the dependence of the membrane capacitance change initiated by a UV flash on the KCl concentration in the electrolyte. The induced pH shift led to a small increase of the BLM capacitance in the absence of Na + ,K + -ATPase containing membrane fragments similarly to that observed earlier [26,27]. The addition of KCl up to 10 mM did not affect this increase.…”

“…The initial capacitance increase, measured immediately after the flash of light, was used for quantitative analysis. Figure 4 shows the dependence of the initial capacitance change as function of the potassium concentration in the presence and absence of magnesium ions (in the latter case, no subsequent relaxation was observed, and this value was measured as the average value after the stepwise shift similarly to that determined earlier [27]). The previously developed theoretical model of competition between proton and sodium or potassium binding was used for quantitative analysis.…”

“…The previously developed theoretical model of competition between proton and sodium or potassium binding was used for quantitative analysis. It allowed the estimation of the binding constants of these ions in the binding site on the cytoplasmic side of Na + ,K + -ATPase [26,27]. According to this model, the dependence of the capacitance change on pH and the concentration of potassium ions is accounted for by Eq.…”

“…Previously, the electrogenic ion transport by the Na + ,K + -ATPase in membrane fragments adsorbed on a bilayer lipid membrane was studied by recording short-circuit currents and changes of the admittance (membrane capacitance and conductivity) induced by rapid photo-activated release of ATP from an inactive form (caged ATP) [23][24][25]. The same method allowed it to study the transport of sodium and potassium ions in exchange for protons by recording changes of the membrane capacitance after a rapid decrease of pH caused by the photo-activated release of protons (caged H + ) [26,27]. This method opens up new possibilities to study binding and transport of potassium ions, which was previously inaccessible by the use of caged ATP.…”